Methods of treating fungal infections using histatin-based peptides

ABSTRACT

Peptides representing defined portions of the amino acid sequences of naturally occurring human and macaque histatins, modified peptides, expression vectors encoding these peptides, and compositions and methods for treatment of fungal infection are described. Some of the histatin-based peptides exhibit superior anti-fungal activity to native, intact histatins. Isolated, naturally occurring macaque histatins and peptide analogs are also described.

GOVERNMENT SUPPORT

The invention described herein was supported in whole or in part byGrant No. DE07652 from the National Institutes of Health, which havecertain rights in the invention.

This application is a division, of application Ser. No. 08/287,717 filedAug. 9, 1994, now U.S. Pat. No. 5,986,503, which is a File Wrappercontinuation of 08/145,030, filed Oct. 28, 1993, which is a File Wrappercontinuation of 07/786,571, filed Nov. 1, 1991, both now abandoned.

BACKGROUND

Infection with the yeast Candida albicans is a prevalent and, in somecases, life-threatening condition affecting otherwise healthy andimmuno-compromised patients. Candidal vaginitis is estimated to affect15 to 55% of healthy young women. Candidal infections often occur indiabetics, during pregnancy, and following medication with antibiotics,steroid hormones, or oral contraceptives. (Tapper-Jones, L. M. et al.(1981) J. Clin. Pathol. 34:706-11; Sobel, J. D. et al. (1984) Infect.Immun. 44:576-580) Oral candidiasis is an early opportunistic infectionof Acquired Immune Deficiency Syndrome (AIDS) in individuals infectedwith human immunodeficiency virus type 1, as well as a complication ofradiation and chemotherapy in cancer patients. (Yeh, C. -K. et al.(1988) J. of Acquired Immune Deficiency Syndromes 1:361-366) Inaddition, candidal infection of denture wearers plays a primary role indental stomatitis, a prevalent oral problem among the elderly. (Pollock,J. J. et al. (1990) NYS Dental J. 56:36-38) Candidal infections of skinand urethra are widespread problems. In patients in intensive care andimmunocompromised patients, systemic fungal infection often leads todeath, since there are few effective anti-fungal pharmaceuticals forintravenous use. (Burnie, J. P. et al. (1985) British Medical Journal290:746-748)

Although several anti-fungal agents are currently available (e.g.,clotrimazole, miconazole, ketoconazole, chlorhexidine, and nystatin),these are not completely effective and can produce adverse side effects.Many are not appropriate for oral or systemic administration. Thus, apotent, naturally occurring anti-fungal substance would provide asignificant improvement in the treatment of fungal infection.

SUMMARY OF THE INVENTION

This invention is based on substantially pure peptides which haveanti-candidal activity greater than that of intact, native histatins.These peptides represent defined portions of the amino acid sequences ofnaturally occurring human and macaque histidine-rich salivary proteinscalled histatins, and will be referred to herein as histatin-basedpeptides. As demonstrated herein, these histatin-based peptides havebeen shown to be superior in anti-candidal activity over the intact,native histatins. Thus, this invention provides compositions fortreatment of fungal infection comprising histatin-based peptides withdefined amino acid sequences. Compositions for anti-fungal treatmentcomprising substantially pure protein having the amino acid sequence ofthe naturally occurring macaque M-histatin 1 are also provided. Proteinpreparations of the macaque histatins, referred to herein as M-histatin1, 2, 3, and 4, are also included. This invention further includescompositions for anti-fungal treatment comprising expression vectorsencoding the histatin-based peptides. Compositions and methods foranti-fungal therapy using bacteria transformed with the above-mentionedexpression vectors for expression of the encoded histatin(s) orhistatin-based peptide(s) in the urinary/reproductive orgastrointestinal tract are also included. Proteins and peptides,expression vectors, transformed cells, and compositions and methods fortreatment of fungal, and in particular, candidal infection using thesesubstances are included within the scope of this invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows the amino acid sequences of human histalin 3, SEQ ID NO: 1and peptides SPM-H3 SEQ ID NO:2, 3P1, SEQ ID NO:3, 3P2, SEQ ID NO:4 3P3,SEQ ID NO:5, 3P4 SEQ ID NO:6, and 3P5 SEQ ID NO:7.

FIG. 2 shows the cationic PAGE electrophoretograms of human (HPS) andmacaque (MPS) parotid saliva protein.

FIG. 3 shows the elution profile of M. fascicularis parotid salivaprotein from a Bio-Gel P-2 column. Inset shows an aliquot of eachfraction (A-F) examined in the cationic PAGE system.

FIG. 4 shows the elution profile of fraction C-F during RP-HPLCchromatography. Inset shows purified preparations of M-histatin 1 andM-histatin 3 subjected to cationic PAGE.

Table 1 shows the amino acid composition of macaque and human histatins.Values in parentheses were the residues identified by automated Edmandegradation for M-histatin 1 and by amino acid analysis for M-histatin3. +Data for these columns are from Oppenheim, F. G. et al. (1988) J.Biol. Chem. 263:7472-7477, which is herein incorporated by reference.

FIG. 5 shows the amino acid sequences of human histatins 1 SEQ ID NO:9and 3 SEQ ID NO:1 compared with that of macaque M-histatin 1. SEQ IDNO:8. Dashed lines indicate gaps. Serines at residue 2 of M-histatin 1and histatin 1 are phosphorylated. Residues in bold-face type indicateamino acid differences.

FIG. 6 shows the amino acid sequences of macaque M-histatin 1 SEQ IDNO:8 and peptides SPM-MHa SEQ ID NO:10, SPM-MHb SEQ ID NO:11, andSPM-MHc SEQ ID NO:12.

Table 2 shows the results of the anti-candidal assays for human histatin3, histatin 3-based peptides, macaque M-histatin 1, and M-histatin1-based peptides.

FIG. 7 shows the interaction of histatin 3 and histatin 3-based peptideswith the cell surface of C. albicans by direct-fluorescence method.Above: Each pair of panels shows phase contrast/fluorescence microscopy,respectively, of: A/B Histatin 3; C/D SPN-H3; E/F SPM-H3; G/H SPC-H3;I/F Control 1; K/L Control 2. Below: Intensity of fluorescence wasscored by inspection. ++++Heavy fluorescence; +++Moderate fluorescence;++Definite fluorescence but minimal in amount; +Barely detectablefluorescence; and-No fluorescence.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to peptides which have anti-fungal activity, inwhich the amino acid sequences represent defined portions of the aminoacid sequences of naturally occurring human and macaque histidine-richsalivary proteins called histatins. (Histatins are also referred to inthe literature as histidine-rich proteins or HRPs.) Histatins are majorsalivary proteins which are synthesized in the parotid andsubmandibular-sublingual secretory glands of humans and Old Worldmonkeys. (Azen, E. A. (1978) Biochem. Genet. 16:79-99) Histatins arebelieved to be part of an extraimmunologic defense system of the oralcavity. The anti-fungal activity of histatins, as well as theirinhibitory effect on several oral bacteria (such as the cariogenicStreptococcus mutans and the periodontal Porphyromonas gingivalis), havebeen demonstrated in vitro. In addition, the observation thatpolyhistidine peptides inactivate herpes simplex virus in vitro and thatwhole saliva contains inhibitors of human immunodeficiency virussuggests the possibility that histatins may have anti-viral activity.These in vitro studies support potential clinical use of compositionscontaining histatins or histatin-based peptides for the treatment oflocal and systemic candidal infection, oral bacterial diseases, such ascaries and periodontitis, and viral infection. (Pollock, J. J. et al.(1984) Infect. Immun. 44:702-7; Oppenheim, F. G. (1986) J. Biol. Chem.261:1177-82; Iacono, V. J. et al. (1983) J. Dent. Res. 63:288; MacKay,B. J. et al. (1984) Infect. Immun. 44:695-701; Xu, T. et al. (1990) J.Dent. Res. 69:239; MacKay, B. J. et al. (1984) J. Dent. Res. 63:288;Oppenheim, F. G. et al. (1988). J. Biol. Chem. 263:7472-7477; Pollock,J. J. et al. (1985) In: Protides of the Biological Fluids: Proceedingsof the Colloquium (H. Peeters, ed.), New York: Pergamon Press, Vol. 32,pp. 309-314; Docherty, J. J. et al. (1987) Anti-microbial Agents andChemotherapy 31(10):1562-1566; Fox, P. C. et al. (1988) JADA116:635-637; Nishikata, M. et al. (199i) Biochem. Biophys. Res. Comm.174 (2): 625-630)

The human histatin proteins have been isolated and sequenced. They havebeen shown to be a family of twelve related low molecular weightproteins. Comparison of the amino acid sequences of the histatinssuggests that histatin 2 and histatins 4-12 may have originated fromspecific proteolytic cleavage of histatin 1 and histatin 3,respectively. (Oppenheim, F. G. et al. (1988) J. Biol. Chem.263:7472-77; Troxler, R. F. et al. (1990) J. Dent. Res. 69(1):2-6)Cloning and sequence analysis of histatin cDNAs further suggest that thehistatins are encoded by two homologous genetic loci, whose primaryproducts are histatins 1 and 3. (Sabatini, L. M. et al. (1989) Biochem.Biophys. Res. Comm. 160:495-502; Vanderspek, J. C. et al. (1990) Arch.Oral Biol. 35(2):137-43)

The amino acid sequences of the anti-fungal peptides of this inventionrepresent defined portions of the amino acid sequence of human histatin3 SEQ ID NO:1 (SPM-H3, SEQ ID NO:2, 3P1 SEQ ID NO:3, 3P2 SEQ ID NO:4,3P4 SEQ ID NO:6, and 3P5SEQ ID NO:87), the complete amino acid sequenceof macaque M-histatin 1 SEQ ID NO:7 protein, or defined portions of theamino acid sequence of M-histatin 1 (SPM-MHa SEQ ID NO:10, SPM-MHb SEQID NO:11, and SPM-MHc SEQ ID NO:12). This is the first time that theentire amino acid sequence of a nonhuman histatin protein is available.The peptides can be obtained from a naturally occurring source ofhistatin, or be produced (e.g., by recombinant DNA techniques or bychemical synthesis) to have the same, or substantially the same, aminoacid sequence as all or a portion of the naturally occurring protein.

The peptides described herein were tested in three assays designed tomeasure separately their effectiveness in killing of blastoconidia, inkilling of germinated cells, and in inhibition of germination of C.albicans. When tested in these assays, the histatin 3-based peptides andM-histatin 1-based peptides were found surprisingly to have superioranti-candidal activity over native, intact histatin 3 and M-histatin 1.In addition, peptides representing all or portions of the amino acidsequences of native histatins which have been modified by, for example,deletion, addition, substitution, or side chain modification of at leastone referred to herein as modified histatin-based peptides. Isolatedpreparations of the macaque histatins, M-histatin 1, 2, 3, and 4, arealso included in this invention.

The following is a description of the histatin-based peptides, themacaque histatins, the anti-fungal activities of the histatin-basedpeptides as measured in assays for killing of Candida blasto-conidia,killing of germinated cells, and inhibition of germination, and modifiedhistatin-based peptides.

Histatin 3-Based Peptides

Histatin 3 was isolated from human parotid salivary secretions. Thehistatin 3-based peptides, SPM-H3, 3P1, 3P2, 3P3, 3P4, and 3P5, werechemically synthesized. The amino acid sequences of histatin 3 SEQ IDNO:1 and its derivative peptides SEQ ID NO:27 are shown in FIG. 1.

Isolation and Characterization of Macaque Histatins

Comparison of the electrophoretic patterns of human and macaque (Macacafascicularis) parotid salivary proteins in a cationic polyacrylamide gelelectrophoresis (PAGE) system revealed the presence of one prominentprotein band (M-histatin 1) with a mobility midway between those ofhuman histatins 1 and 3 (FIG. 2). In addition, three minor bands werealso noted (M-histatins 2, 3, and 4). M-histatin 2 was close to themajor band, while M-histatins 3 and 4 represent a second pair ofproteins migrating more cathodically and exhibiting mobilities midwaybetween those of histatins 3 and 5.

Isolated preparations of M-histatins 1 and 3 were made, as illustratedin FIGS. 3 and 4. Isolation of M-histatin 2 and 4 proteins would followessentially the same procedure.

The amino acid composition of M-histatin 1 and M-histatin 3 was comparedwith that of human histatins 1 and 3 in Table 1. M-histatin 1 containsten residues of histidine, five of arginine, four of glycine andtyrosine, and three of aspartic acid/asparagine, glutamicacid/glutamine, serine, and lysine, and lacks threonine, proline,alanine, valine, cysteine, methionine, and isoleucine. The amino acidcomposition of M-histatin 3 is similar to that of M-histatin 1 in thatit is enriched with respect to histidine, arginine, and glycine, butlacks leucine. The amino acid compositions of both M-histatins 1 and 3are similar to those of human histatins 1 and 3, respectively.

The complete amino acid sequence of M-histatin 1 was determined SEQ IDNO:8. The protein has a pI of 8.5 and contains 38 amino acid residues.It has a molecular weight of 4881.8, with 26.3% consisting of histidineby weight. The hydropathicity plot of M-histatin 1 indicates that themolecule is hydrophilic along its entire length, and a prediction ofsecondary structure indicates that the molecule is devoid ofalpha-helices and beta sheets, but instead contains a series of betaturns that would tend to give the protein a random structure in aqueoussolutions. The experimentally determined sequence is in completeagreement with the amino acid composition of the protein (Table 1).

The amino acid composition of M-histatin 3 is nearly identical to thatof the N-terminal 20 amino acid residues of M-histatin 1. This suggeststhat M-histatin 3 may arise from M-histatin 1 by chymotryptic-likecleavage between Phe₂₀ and His₂₁.

The primary structures of M-histatin 1 SEQ ID NO:8 and human histatins 1SEQ ID NO:9 and 3 SEQ ID NO:1 were compared, with gaps introduced fordelineation of inserts that are unique to one, but not the other, ofthese proteins (FIG. 5). M-histatin 1 contains a six-residue sequence,-Arg-His-Gly-His-His-Lys- (residues 10-15 SEQ ID NO:13), which is absentfrom histatins 1 and 3, and histatin 1 contains a hexapeptide sequence,-Phe-Pro-Phe-Tyr-Gly-Asp- (residues 24-29 SEQ ID NO:14), which is notpresent in either M-histatin 1 or histatin 3. Otherwise, these proteinsare remarkably similar. M-histatin 1 and histatin 1 exhibit 89% sequencesimilarity, M-histatin 1 and histatin 3 exhibit 91% sequence similarity,and histatins 1 and 3 exhibit 88% sequence similarity, if thehexapeptide sequences unique to M-histatin 1 and histatin 1 SEQ. ID NO:13 and 14, respectively, are not considered.

M-Histatin 1-Based Peptides

Peptides representing portions of the amino acid sequence of M-histatin1 were chemically synthesized. The amino acid sequences of M-histatin 1SEQ ID NO:8 and the M-histatin 1-based peptides, SPM-MHa SEQ ID NO:10,SPM-MHb SEQ ID NO:11, and SPM-MHc SEQ ID NO:12, are shown in FIG. 6.

Anti-Fungal Activities of Histatin 3 and M-Histatin 1 and TheirDerivative Peptides

C. albicans is a dimorphic yeast. It can exist in a yeast orblastoconidial form, which upon germination develops into the hyphal orgerminated form. While the germinated form is considered to be moreinvasive, most of the C. albicans isolates harvested from the oralcavities of healthy individuals appear to be in the blastoconidial form.(Arendorf, T. M. et al. (1980) Arch. Oral Biol. 25:1-10; Gow, N. A. R.et al. (1987) Criti. Rev. Microbiol. 15:73-78; Odds, F. C. (1988)Candida and Candidosis, 2nd ed., Bailliere Tindall, London, England)Anti-fungal activity of histatin 3, M-histatin 1, and peptides based onportions of their amino acid sequences was measured in assays designedto test separately the effectiveness of the proteins against alldevelopmental forms of Candida (Table 2). These assays, which measurekilling of blastoconidia, killing of germinated cells, and inhibition ofgermination of C. albicans are described in Xu et al., which is hereinincorporated by reference. (Xu, T. et al. (1991) Infect. Immun.59(8):2549-2554). SPM-H3 was found to be about 5-10X more potent thanhistatin 3 in all three assays, demonstrating its superiority to thenative, intact protein in both fungicidal and fungistatic activities.3P1 and 3P3 were about 6-30X more potent than histatin 3 in thefungicidal activities, but had only about 1-2X the fungistatic activityof histatin 3. 3P2 had fungicidal activities similar to 3P1 and 3P3, butvirtually no fungistatic activity. 3P4 was less potent than 3P1, 3P2,and 3P3 in fungicidal activities (about 2X more potent than histatin 3),and was also ineffective in inhibiting germination. 3P5, like 3P4, hadabout 2X the anti-fungal activity of histatin 3 in killing ofbiastoconidia and no fungistatic activity, but was about 5X more potentthan 3P4 in killing of germinated cells.

Macaque M-histatin 1 was comparable in all three activities to humanhistatin 3. All three M-histatin 1-based peptides were about 2-5X moreeffective in fungicidal activities, but less potent in inhibition ofgermination than the intact macaque histatin. These results suggest thata combination of histatin-based peptides may be used for maximumanti-fungal activity against Candida in all three stages of its lifecycle.

Comparison of the anti-candidal activities of the synthetic peptideswithin each group (histatin 3 or M-histatin 1) does not indicate asimple correlation between anti-fungal effectiveness, as measured by thethree assays, and any single aspect of the primary structure of thepeptides, that is, presence of N-terminal or C-terminal residues whichmay indicate functional domains, peptide length, or content of the basicamino acids, histidine, lysine, and arginine (Table 2). Thus, theanti-fungal potency of a histatin-based peptide seems to reside in acombination of the particular amino acid sequence and the length, thatis, in the defined sequence of the peptide.

The mode by which histatins and histatin-based peptides exert theiranti-fungal activity on C. albicans is suggested by preliminary resultsof a binding assay of labeled peptide portions of histatin 3 to Candidacells (FIG. 7). Synthetic peptides corresponding to the N-terminalportion (SPN-H3, residues 1-11), middle portion (SPM-H3, residues12-25), and C-terminal portion (SPC-H3, residues 23-32) of the histatin3 amino acid sequence were labeled with a fluorescent dye and incubatedwith Candida yeast and germinated forms. Binding was detected byfluorescence microscopy. Of the three peptide portions, only SPM-H3bound significantly to Candida cells, and only SPM-H3 exhibitedsignificant anti-candidal activity (Table 2). That the binding of SPM-H3was specific was indicated by inhibition of binding of labeled SPM-H3 bypreincubation of Candida cells with unlabeled SPM-H3. These resultsindicate that SPM-H3 interacts directly at the surface of Candida yeastand germinated cells.

Other Anti-Fungal Histatin-Based Peptides Including Modified Peptides

The analysis of the synthetic peptides suggests that optimization of thedefined portion of the amino acid sequence of a naturally occurringhistatin which gives maximal anti-fungal activity and optimization bymodification of a histatin-based peptide are best done empirically. Thatis, peptides must be produced somewhat randomly and then tested inassays such as those described herein. Modified histatin-based peptidescan differ from the corresponding naturally-occurring sequence byaddition, deletion, substitution, or side-chain modification of at leastone amino acid, or other chemical modification.

Peptide sequences containing permutations of the amino acid sequences ofnative histatins and modified peptides can be produced by known methods,such as recombinant DNA techniques and solid-phase synthesis. Cloned DNAencoding the human histatins may be obtained as described by Sabatini etal. or Vanderspek et al., whose teachings are incorporated herein byreference. (Sabatini. L. M. et al. (1989) Biochem. Biophys. Res. Comm.160:495-502; Vanderspek, J. C. et al. (1990) Arch. Oral Biol.35(2):137-43) cDNA encoding the macaque M-histatin 1 protein can becloned by recombinant DNA techniques, for instance, by using degenerateoligonucleotides based on the amino acid sequence of the histatin asprimers for polymerase chain reaction amplification. Alternatively,oligonucleotides encoding histatins or histatin-based peptides can besynthesized chemically using commercially available equipment. They canthen be made double-stranded and cloned into vectors for amplificationin prokaryotic or eukaryotic host cells.

Histatin-based peptides can be produced in a variety of expressionvector/host systems, which are available commercially or can bereproduced according to recombinant DNA and cell culture techniques. Thevector/host expression systems can be prokaryotic or eucaryotic, and caninclude bacterial, yeast, insect, mammalian, and viral expressionsystems. The construction of expression vectors encoding histatin-basedpeptides, transfer of the vectors into various host cells, andproduction of peptides from transformed host cells can be accomplishedusing genetic engineering techniques, as described in manuals such asMolecular Cloning and Current Protocols in Molecular Biology, whoseteachings are incorporated herein by reference. (Sambrook, J., Fritsch,E. F. and Maniatis, T. (1989) Molecular Cloning, Second Edition, ColdSpring Harbor Laboratory Press; Ausubel, F. M. et al., eds., CurrentProtocols in Molecular Biology, New York: Greene Publishing Associatesand Wiley-Interscience)

Modified histatin based peptides can be synthesized chemically, or beproduced from cloned DNAs containing mutated nucleotide sequences.Histatin-based peptides encoded by expression vectors may be modifieddue to post-translational processing in a particular expressionvector/host cell system. (See, e.g., Wold, F. (1981) Ann. Rev. Biochem.50:783-814) Histatin-based peptides may also be modified by chemicalalteration of amino acid side-chain groups, or by other covalentmodification. (See, e.g., Glazer, A. N. et al. (1975) ChemicalModification of Proteins, North Holland; Katre, N. V. et al. (1987)Proc. Natl. Acad. Sci. USA 84:1487)

Therapeutic Applications

The histatin-based peptides of this invention, representing definedportions of the amino acid sequence of human histatin 3 SEQ. ID NO: 1SPM-H3 SEQ. ID NO: 2 3P1, SEQ ID NO: 3 3P2, SEQ ID NO: 4, 3P3 SEQ IDNO:5 3P4 SEQ ID NO:6, and 3P5 SEQ ID NO:7, or all or defined portions ofthe amino acid sequence of M-histatin 1: M-histatin 1, SPM-MHa SEQ IDNO:10, SPM-MHb SEQ ID NO:11, and SPM-MHc SEQ ID NO: 12, can be used incompositions and methods of treatment for fungal, and in particular,candidal infection. These methods of treatment for fungal infectionapply to preventive treatment as well. The compositions may containcombinations of histatin-based peptides, in order to obtain maximumactivity against all developmental forms of Candida. The ionic strength,presence of various mono- and divalent ions, and pH of the compositionsmay be adjusted to obtain maximum anti-fungal activity of thehistatin-based peptides, as described in Xu et al. (Xu, T. et al. (1991)Infect. Immun. 59(8):2549-54) Carriers appropriate for administration ofanti-fungal agents to the vagina, the urethra, the oral cavity, and skinare known, and described, for instance, in U.S. Pat. No. 4,725,576(Fungicidal Polypeptide Compositions Containing L-His and Methods forUse Therefore by J. J. Pollock and B. J. MacKay, Feb. 16, 1988).Compositions for treatment of systemic infection can be administered byvarious routes, such as intravenously or subdermally.

Expression vectors encoding the above-mentioned peptides can be used incompositions and methods for anti-fungal treatment. Expression vectorsmay be administered in compositions which introduce genetic materialencoding histatin-based peptides into cells of the patients. Forexample, recombinant expression vectors based on retroviruses oradenovirus vaccines may be used to infect patients.

A method of anti-fungal therapy using the above-described expressionvectors is bacterial substitution therapy. Bacterial substitutiontherapy can be used to treat fungal infection of areas in theurinary/reproductive and/or gastrointestinal tracts of a patient. Thetherapy comprises the following: 1) transforming bacteria with DNAcomprising an expression vector which encodes a histatin-based peptidedescribed above, thereby producing transformed cells; 2) selectingtransformed cells which express the peptide encoded by the expressionvector, thereby obtaining tranformed cells which express ahistatin-based peptide; and 3) administering transformed cells whichexpress a histatin-based peptide in an appropriate carrier to theinfected area.

One application of bacterial substitution therapy is treatment of fungalinfections of the oral cavity. A number of species of the oral bacterialStreptococcus can be used as vehicles for the expression vectors. Forexample, recombinant S. lactis has been used in oral immunization ofmice against a heterologous antigen. (Iwaki, M. et al. (1990) Infect.Immun. 58(9):2929-34) Other oral bacteria which can be used as vehiclesfor the expression vectors, plasmids for constructing expression vectorscapable of amplification in oral bacterial host cells, transformationmethods, and administration of compositions containing oral bacteria tohumans have been described. (See, e.g., Kuramitsu, H. K. et al. (1984)J. General Microbiology 130:2497-2500; LeBlanc, D. J. et al. (1978)Proc. Natl. Acad. Sci. USA 75(7):3484-3487; Macrina, F. L. et al. (1980)J. Bacteriology 143(3):1425-1435; Kuramitsu, H. K. et al. (1982) Infect.Immun. 36(1):435-436; Svanberg, M. et al. (1984) Infect. Immun.43(3):817-821).

The compositions and methods for treatment of fungal infectionsdiscussed above are not limited to use in humans, but can haveveterinary applications as well.

Furthermore, the above-described compositions and methods for treatmentof fungal infection can also be used for treatment of bacterialinfections (e.g., of S . mutans or P. gingivalis) and viral infections(e.g., of herpex simplex virus or human immunodeficiency virus type 1).

Examples

1. Chemical synthesis of Histatin-Based Peptides

Isolation and amino acid sequence of human histatin 3 SEQ ID NO: 1 aredescribed in Oppenheim et al., whose teachings are herein incorporatedby reference. (Oppenheim, F. G. et al. (1988) J. Biol. Chem.263(16):7472-7477) Human and macaque histatin-based peptides weresynthesized by the solid phase method of Merrifield. (Merrifield, B.(1986.) Science 232:341-47) Peptides were synthesized by aMilliGen/Bioresearch Sam-Two Peptide Synthesizer using Fmoc L-amino acidkits (Millipore, Bedford, Mass.) and purified on a TSK ODS-120T C₁₈column (5 μm, 4.6×250 mm) using RP-HPLC (Pharmacia-LKB). The purifiedpeptides were quantified by amino acid analysis on a Beckman System 6300amino acid analyzer.

3. Candida Binding Assay

Histatin 3 and synthetic peptides were labeled with a fluorochrome(5-(and 6)-carboxyfluorescein, succinimidyl ester) (Molecular Probes,Eugene, Or.) according to the method of Forni and Perters. (Forni, L.and Perters, S. (1984) Methods Enzymol. 108:413-425) Severalmodifications have been made. Briefly, histatin and peptides in 0.1Msodium bicarbonate buffer (pH 9.0) were incubated with the fluorochrome(freshly prepared in dimethyl sulfoxide) for 24 hours at 4° C. in thedark, respectively. Reaction was stopped by adding 50 mM glycine in 0.1MTris-HCl and incubated for 3 hours at 4° C. in the dark. Theconjugated-protein/peptide was separated from unbound dye and freeprotein/peptides by RP-HPLC. The amount of the conjugates was determinedby amino acid analysis. The initial protein/peptide vs. fluorochrome dyeratio was 1:8 (μmol/μmol). The conjugate percentage was about 90% byusing the modified method.

C. albicans (2×10⁷ cells/ml, in PBS) in yeast or germinated forms wereincubated with conjugated histatin or peptides (0.5-1.0 nmol/mlconcentration) for 30 minutes at 25° C. in 1.5 ml test tubes,respectively. The tubes were centrifuged three times at 1000 g for 5minutes. After discarding the supernatant, the tubes were refilled with1 ml PBS to suspend the cell pellet. Cell suspension (200 μl) was addedinto a chamber slide and incubated at 25° C. for 30 minutes in the dark.After washing with PBS and removal of the chamber, the slide wasmounted, observed (at 450-490 nm) and photographed as described above.The controls were processed with unconjugated protein/peptides (0.5-1.0nmol/ml) and fluorochrome (2 μg/ml), respectively.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 14                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       AspSerHisAlaLysArgHisHisGlyTyrLysArgLysPheHisGlu                              151015                                                                        LysHisHisSerHisArgGlyTyrArgSerAsnTyrLeuTyrAspAsn                              202530                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ArgLysPheHisGluLysHisHisSerHisArgGlyTyrArg                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LysArgHisHisGlyTyrLysArgLysPheHisGluLysHisHisSer                              151015                                                                        HisArgGlyTyrArg                                                               20                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlyTyrLysArgLysPheHisGluLysHisHisSerHisArgGlyTyr                              151015                                                                        Arg                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LysArgHisHisGlyTyrLysArgLysPheHisGluLysHisHisSer                              151015                                                                        HisArg                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlyTyrLysArgLysPheHisGluLysHisHisSerHisArg                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LysArgHisHisGlyTyrLysArgLysPheHisGluLysHisHis                                 151015                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AspSerHisGluGluArgHisHisGlyArgHisGlyHisHisLysTyr                              151015                                                                        GlyArgLysPheHisGluLysHisHisSerHisArgGlyTyrArgSer                              202530                                                                        AsnTyrLeuTyrAspAsn                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AspSerHisGluLysArgHisHisGlyTyrArgArgLysPheHisGlu                              151015                                                                        LysHisHisSerHisArgGluPheProPheTyrGlyAspTyrGlySer                              202530                                                                        AsnTyrLeuTyrAspAsn                                                            35                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ArgHisGlyHisHisLysTyrGlyArgLysPheHisGluLysHisHis                              151015                                                                        SerHisArgGlyTyrArg                                                            20                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      ArgHisGlyHisHisLysTyrGlyArgLysPheHisGluLysHisHis                              151015                                                                        SerHisArg                                                                     (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      ArgHisGlyHisHisLysTyrGlyArgLysPheHisGluLysHisHis                              151015                                                                        (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ArgHisGlyHisHisLys                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      PheProPheTyrGlyAsp                                                            15                                                                            __________________________________________________________________________

We claim:
 1. A method for treating fungal infection comprisingadministering a composition comprising a substantially pure peptidehaving an amino acid sequence selected from the group consisting of:a)the amino acid sequence of SPM-H3 as set forth in SEQ ID NO: 2; b) theamino acid sequence of 3P1 as set forth in SEQ ID NO: 3; c) the aminoacid sequence of 3P2 as set forth in SEQ ID NO: 4; d) the amino acidsequence of 3P3 as set forth in SEQ ID NO: 5; e) the amino acid sequenceof 3P4 as set forth in SEQ ID NO: 6; f) the amino acid sequence of 3P5as set forth in SEQ ID No: 7; g) the amino acid sequence of M-histatin 1as set forth in SEQ ID NO: 8; h) the amino acid sequence of SPM-MHa asset forth in SEQ ID NO: 10; i) the amino acid sequence of SPM-MHb as setforth in SEQ ID NO: 11; j) the amino acid sequence of SPM-MHc as setforth in SEQ ID NO: 12; and k) combinations of two or more of the above.2. A method for treating candidal infection comprising administering acomposition of claim
 1. 3. A method for treating fungal infectioncomprising administering a composition of claim 1, wherein the fungalinfection is an infection selected from the group consisting of:a) aninfection of the oral cavity; b) an infection of the vagina; c) aninfection of the urethra; d) an infection of the skin; and e) a systemicinfection.